Transition metal complexes with (pyridyl)imidazole ligands

ABSTRACT

Novel transition metal complexes of iron, cobalt, ruthenium, osmium, and vanadium are described. The transition metal complexes can be used as redox mediators in enzyme-based electrochemical sensors. The transition metal complexes include substituted or unsubstituted (pyridyl)imidazole ligands. Transition metal complexes attached to polymeric backbones are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/269,690, filed on Nov. 12, 2008, now U.S. Pat. No. 7,615,637, whichis a continuation of U.S. patent application Ser. No. 11/361,427 filedon Feb. 24, 2006, now U.S. Pat. No. 7,465,796, which is a continuationof U.S. patent application Ser. No. 10/714,835, filed on Nov. 14, 2003,now U.S. Pat. No. 7,074,308, which is a continuation of U.S. patentapplication Ser. No. 10/143,300 filed on May 9, 2002, now U.S. Pat. No.6,676,816, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/290,537 of Fei Mao, filed on May 11, 2001 andentitled “Transition Metal Complexes with (Pyridyl)imidazole Ligands”,the disclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to transition metal complexes with(pyridyl)imidazole ligands. In addition, the invention relates to thepreparation of the transition metal complexes and to the use of thetransition metal complexes as redox mediators.

BACKGROUND OF THE INVENTION

Enzyme-based electrochemical sensors are widely used in the detection ofanalytes in clinical, environmental, agricultural and biotechnologicalapplications. Analytes that can be measured in clinical assays of fluidsof the human body include, for example, glucose, lactate, cholesterol,bilirubin and amino acids. Levels of these analytes in biologicalfluids, such as blood, are important for the diagnosis and themonitoring of diseases.

Electrochemical assays are typically performed in cells with two orthree electrodes, including at least one measuring or working electrodeand one reference electrode. In three electrode systems, the thirdelectrode is a counter-electrode. In two electrode systems, thereference electrode also serves as the counter-electrode. The electrodesare connected through a circuit, such as a potentiostat. The measuringor working electrode is a non-corroding carbon or metal conductor. Uponpassage of a current through the working electrode, a redox enzyme iselectrooxidized or electroreduced. The enzyme is specific to the analyteto be detected, or to a product of the analyte. The turnover rate of theenzyme is typically related preferably, but not necessarily, linearly)to the concentration of the analyte itself, or to its product, in thetest solution.

The electrooxidation or electroreduction of the enzyme is oftenfacilitated by the presence of a redox mediator in the solution or onthe electrode. The redox mediator assists in the electricalcommunication between the working electrode and the enzyme. The redoxmediator can be dissolved in the fluid to be analyzed, which is inelectrolytic contact with the electrodes, or can be applied within acoating on the working electrode in electrolytic contact with theanalyzed solution. The coating is preferably not soluble in water,though it may swell in water. Useful devices can be made, for example,by coating an electrode with a film that includes a redox mediator andan enzyme where the enzyme is catalytically specific to the desiredanalyte, or its product. In contrast to a coated redox mediator, adiffusional redox mediator, which can be soluble or insoluble in water,functions by shuttling electrons between, for example, the enzyme andthe electrode. In any case, when the substrate of the enzyme iselectrooxidized, the redox mediator transports electrons from thesubstrate-reduced enzyme to the electrode; and when the substrate iselectroreduced, the redox mediator transports electrons from theelectrode to the substrate-oxidized enzyme.

Recent enzyme-based electrochemical sensors have employed a number ofdifferent redox mediators such as monomeric ferrocenes, quinoidcompounds including quinines (e.g., benzoquinones), nickel cyclamates,and ruthenium amines. For the most part, these redox mediators have oneor more of the following limitations: the solubility of the redoxmediators in the test solutions is low, their chemical, light, thermal,and/or pH stability is poor, or they do not exchange electrons rapidlyenough with the enzyme or the electrode or both. Some mediators withadvantageous properties are difficult to synthesize. Additionally, theredox potentials of some of these reported redox mediators are sooxidizing that at the potential at which the reduced mediator iselectrooxidized on the electrode, solution components other than theanalyte are also electrooxidized. Some other of these reported redoxmediators are so reducing that solution components, such as, forexample, dissolved oxygen, are also rapidly electroreduced. As a result,the sensor utilizing the mediator is not sufficiently specific.

SUMMARY OF THE INVENTION

The present invention is directed to novel transition metal complexes.The present invention is also directed to the use of the complexes asredox mediators. The preferred redox mediators typically exchangeelectrons rapidly with enzymes and electrodes, are stable, can bereadily synthesized, and have a redox potential that is tailored for theelectrooxidation of analytes, such as glucose for example.

One embodiment of the invention is a transition metal complex having thegeneral formula set forth below.

In this general formula, M is cobalt, iron, ruthenium, osmium, orvanadium; c is an integer selected from −1 to −5, 0, or +1 to +5indicating a positive, neutral, or negative charge; X represents atleast one counter ion; d is an integer from 0 to 5 representing thenumber of counter ions, X; L and L′ are independently selected from thegroup consisting of:

and L₁ and L₂ are other ligands. In the formula for L and L′, R′₁, is asubstituted or an unsubstituted alkyl, alkenyl, or aryl group.Generally, R′₃, R′₄, R_(a), R_(b), R_(d), and R_(d) are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂,or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino,alkylthio, alkenyl, aryl, or alkyl.

The transition metal complexes of the present invention are effectivelyemployed as redox mediators in electrochemical sensors, given their veryfast kinetics. More particularly, when a transition metal complex ofthis invention is so employed, rapid electron exchange between thetransition metal complex and the enzyme and/or the working electrode inthe sensor device occurs. This electron exchange is sufficiently rapidto facilitate the transfer of electrons to the working electrode thatmight otherwise be transferred to another electron scavenger in thesystem. The fast kinetics of the mediator is generally enhanced when L₂of a mediator of the formula provided above is a negatively chargedligand.

The transition metal complexes of the present invention are also quitestable. For example, when such a complex is used as a mediator in anelectrochemical sensor, the chemical stability is generally such thatthe predominant reactions in which the mediator participates are theelectron-transfer reaction between the mediator and the enzyme and theelectrochemical redox reaction at the working electrode. The chemicalstability may be enhanced when a mediator of the formula provided above,wherein L₂ is a negatively charged ligand, has a “bulky” chemicalligand, L₁, that shields the redox center, M, and thereby reducesundesirable chemical reactivity beyond the desired electrochemicalactivity.

The electrochemical stability of the transition metal complexes of thepresent invention is also quite desirable. For example, when such acomplex is used as a mediator in an electrochemical sensor, the mediatoris able to operate in a range of redox potentials at whichelectrochemical activity of common interfering species is minimized andgood kinetic activity of the mediator is maintained.

Thus, the present invention provides novel transition metal complexesthat are particularly useful as redox mediators in electrochemicalsensing applications. The advantageous properties and characteristics ofthe transition metal complexes of the present invention make them idealcandidates for use in the electrochemical sensing of glucose, anapplication of particular importance in the treatment of diabetes inhuman populations.

DETAILED DESCRIPTION

When used herein, the definitions set forth below in quotations definethe stated term.

The term “alkyl” includes linear or branched, saturated aliphatichydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, theterm “alkyl” includes both alkyl and cycloalkyl groups.

The term “alkoxy” describes an alkyl group joined to the remainder ofthe structure by an oxygen atom. Examples of alkoxy groups includemethoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, and thelike. In addition, unless otherwise noted, the term ‘alkoxy’ includesboth alkoxy and cycloalkoxy groups.

The term “alkenyl” describes an unsaturated, linear or branchedaliphatic hydrocarbon having at least one carbon-carbon double bond.Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 2-methyl-1-propenyl, and the like.

A “reactive group” is a fractional group of a molecule that is capableof reacting with another compound to couple at least a portion of thatother compound to the molecule. Reactive groups include carboxy,activated ester, sulfonyl halide, sulfonate ester, isocyanate,isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine,acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine,alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate,halotriazine, imido ester, maleimide, hydrazide, hydroxy, andphoto-reactive azido aryl groups. Activated esters, as understood in theart, generally include esters of succinimidyl, benzotriazolyl, or arylsubstituted by electron-withdrawing groups such as sulfo, nitro, cyano,or halo groups; or carboxylic acids activated by carbodiimides.

A “substituted” functional group (e.g., substituted alkyl, alkenyl, oralkoxy group) includes at least one substituent selected from thefollowing: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH₂, alkylamino,dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido,hydrazino, alkylthio, alkenyl, and reactive groups.

A “biological fluid” is any body fluid or body fluid derivative in whichthe analyte can be measured, for example, blood, interstitial fluid,plasma, dermal fluid, sweat, and tears.

An “electrochemical sensor” is a device configured to detect thepresence of or measure the concentration or amount of an analyte in asample via electrochemical oxidation or reduction reactions. Thesereactions typically can be transduced to an electrical signal that canbe correlated to an amount or concentration of analyte.

A “redox mediator” is an electron transfer agent for carrying electronsbetween an analyte or an analyte-reduced or analyte-oxidized enzyme andan electrode, either directly, or via one or more additional electrontransfer agents. Redox mediators that include a polymeric backbone mayalso be referred to as “redox polymers”.

“Electrolysis” is the electrooxidation or electroreduction of a compoundeither directly at an electrode or via one or more electron transferagents (e.g., redox mediators or enzymes).

The term “reference electrode” includes both a) reference electrodes andb) reference electrodes that also function as counter electrodes (i.e.,counter/reference electrodes), unless otherwise indicated.

The term “counter electrode” includes both a) counter electrodes and b)counter electrodes that also function as reference electrodes, (i.e.,counter/reference electrodes), unless otherwise indicated.

Generally, the present invention relates to transition metal complexesof iron, cobalt, ruthenium, osmium, and vanadium having(pyridyl)imidazole ligands. The invention also relates to thepreparation of the transition metal complexes and to the use of thetransition metal complexes as redox mediators. In at least someinstances, the transition metal complexes have one or more of thefollowing characteristics: redox potentials in a particular range, theability to exchange electrons rapidly with electrodes, the ability torapidly transfer electrons to or rapidly accept electrons from an enzymeto accelerate the kinetics of electrooxidation or electroreduction of ananalyte in the presence of an enzyme or another analyte-specific redoxcatalyst. For example, a redox mediator may accelerate theelectrooxidation of glucose in the presence of glucose oxidase orPQQ-glucose dehydrogenase, a process that can be useful for theselective assay of glucose in the presence of other electrochemicallyoxidizable species. Some embodiments of the invention may be easier ormore cost-effective to make synthetically or use more widely availableor more cost-effective reagents in synthesis than other transition metalredox mediators.

Compounds having Formula 1, set forth below, are examples of transitionmetal complexes of the present invention.

M is a transition metal and is typically iron, cobalt, ruthenium,osmium, or vanadium. Ruthenium and osmium are particularly suitable forredox mediators.

L and L′ are each bidentate, substituted or unsubstituted2-(2-pyridyl)imidazole ligands having the Structure 2 set forth below.

In Structure 2, R′₁ is a substituted or an unsubstituted aryl, alkenyl,or alkyl. Generally, R′₁ is a substituted or an unsubstituted C1-C12alkyl or alkenyl, or an aryl, such as phenyl, optionally substitutedwith a substituent selected from a group consisting of —Cl, —F, —CN,amino, carboxy, C1-C6 alkyl, C1-C6 alkylthio, C1-C6 alkylamino, C1-C6dialkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkoxy, C1-C6alkoxycarbonyl, and C1-C6 alkylcarboxamido. R′₁ is typically methyl or aC1-C12 alkyl that is optionally substituted with a reactive group, or anaryl optionally substituted with C1-C2 alkyl, C1-C2 alkoxy, —Cl, or —F.

Generally, R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂,substituted or unsubstituted alkoxylcarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino,alkylthio, alkenyl, aryl, or alkyl. Alternatively, R_(c) and R_(d) incombination and/or R′₃ and R′₄ in combination can form a saturated orunsaturated 5- or 6-membered ring. Typically, the alkyl and alkoxyportions are C1 to C12. The alkyl or aryl portions of any of thesubstituents are optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. Generally, R′₃, R′₄, R_(a), R_(b),R_(c) and R_(d) are independently —H or unsubstituted alkyl groups.Typically, R_(a) and R_(c) are —H and R′₃, R′₄, R_(b), and R_(d) are —Hor methyl.

Preferably, the L and L′ ligands are the same. Herein, references to Land L′ may be used interchangeably.

In Formula 1, c is an integer indicating the charge of the complex.Generally, c is an integer selected from −1 to −5 or +1 to +5 indicatinga positive or negative charge or 0 indicating a neutral charge. For anumber of osmium complexes, c is +1, +2, or +3.

X represents counter ion(s). Examples of suitable counter ions includeanions, such as halide (e.g., fluoride, chloride, bromide or iodide),sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, andcations (preferably, monovalent cations), such as lithium, sodium,potassium, tetralkylammonium, and ammonium. Preferably, X is a halide,such as chloride. The counter ions represented by X are not necessarilyall the same.

d represents the number of counter ions and is typically from 0 to 5.

L₁ and L₂ are ligands attached to the transition metal via acoordinative bond. L₁ and L₂ are monodentate ligands, at least one ofwhich is a negatively charged monodentate ligand. While L₁ and L₂ may beused interchangeably, L₂ is generally referred to as a negativelycharged ligand merely by way of convenience. Herein, the term“negatively charged ligand” is defined as a ligand in which thecoordinating atom itself is negatively charged so that on coordinationto a positively charged metal, the negative charge is neutralized. Forexample, a halide such as chloride or fluoride meets the presentdefinition while a pyridine ligand bearing a negatively chargedsulfonate group does not because the sulfonate group does notparticipate in coordination. Examples of negatively charged ligandsinclude, but are not limited to, —F, —Cl, —Br, —I, —CN, —SCN, —OH,alkoxy, alkylthio, and phenoxide. Typically, the negatively chargedmonodentate ligand is a halide.

Examples of other suitable monodentate ligands include, but are notlimited to, H₂O, NH₃, alkylamine, dialkylamine, trialkylamine, orheterocyclic compounds. The alkyl or aryl portions of any of the ligandsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. Any alkyl portions of themonodentate ligands generally contain 1 to 12 carbons. More typically,the alkyl portions contain 1 to 6 carbons. In other embodiments, themonodentate ligands are heterocyclic compounds containing at least onenitrogen, oxygen, or sulfur atom. Examples of suitable heterocyclicmonodentate ligands include imidazole, pyrazole, oxazole, thiazole,triazole, pyridine, pyrazine and derivatives thereof. Suitableheterocyclic monodentate ligands include substituted and unsubstitutedimidazole and substituted and unsubstituted pyridine having the generalFormulas 3 and 4, respectively, as set forth below.

With regard to Formula 3, R₇ is generally a substituted or unsubstitutedalkyl, alkenyl, or aryl group. Generally, R₇ is a substituted orunsubstituted C1 to C12 alkyl or alkenyl, or an aryl, such as phenyl,optionally substituted with a substituent selected from a groupconsisting of —Cl, —F, —CN, amino, carboxy, C1-C6 alkyl, C1-C6alkylthio, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6alkylaminocarbonyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, and C1-C6alkylcarboxamido. R₇ is typically methyl or a C1-C12 alkyl that isoptionally substituted with a reactive group, or an aryl optionallysubstituted with C1-C2 alkyl, C1-C2 alkoxy, —Cl, or —F.

Generally, R₈, R₉ and R₁₀ are independently —H, —F, —Cl, —Br, —I, —NO₂,—CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R₉ and R₁₀, in combination, form a fused 5- or 6-memberedring that is saturated or unsaturated. The alkyl portions of thesubstituents generally contain 1 to 12 carbons and typically contain 1to 6 carbon atoms. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. In some embodiments, R₈, R₉ andR₁₀ are —H or substituted or unsubstituted alkyl. Preferably, R₈, R₉ andR₁₀ are —H.

With regard to Formula 4, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —OH, —NH₂, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Thealkyl or aryl portions of any of the substituents are optionallysubstituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Generally, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are —H, methyl,C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 loweralkyl substituted with a reactive group.

One example includes R₁₁ and R₁₅ as —H, R₁₂ and R₁₄ as the same and —Hor methyl, and R₁₃ as —H, C1 to C12 alkoxy, —NH₂, C1 to C12 alkylamino,C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino,hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12alkyl. The alkyl or aryl portions of any of the substituents areoptionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group.

Examples of suitable transition metal complexes include[Os[1-methyl-2-(2-pyridyl)imidazole]₂(1-methylimidazole)Cl]²⁺2Cl— (alsowritten as [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—) where L₁ is

L₂ is Cl; c is +2; d is 2; X is Cl—, and L and L′ are

The transition metal complexes of Formula 1 also include transitionmetal complexes that are coupled to a polymeric backbone through one ormore of L, L′, L₁, and L₂. In some embodiments, the polymeric backbonehas at least one functional group that acts as a ligand of thetransition metal complex. Such polymeric backbones include, for example,poly(4-vinylpyridine) and poly(N-vinylimidazole) in which the pyridineand imidazole groups, respectively, can act as monodentate ligands ofthe transition metal complex. In other embodiments, the transition metalcomplex can be the reaction product between a reactive group on aprecursor polymer and a reactive group on a ligand of a precursortransition metal complex (such as complex of Formula 1 where one of L,L′, L₁, and L₂ includes a reactive group, as described above). Suitableprecursor polymers include, for example, poly(acrylic acid) (Formula 7),styrene/maleic anhydride copolymer (Formula 8), methylvinylether/maleicanhydride copolymer (GANTREZ polymer) (Formula 9),poly(vinylbenzylchloride) (Formula 10), poly(allylamine) (Formula 11),polylysine (Formula 12), carboxy-poly(vinylpyridine) (Formula 13), andpoly(sodium 4-styrene sulfonate) (Formula 14). The numbers n, n′ and n″appearing variously in these formulas may vary widely. Merely by way ofexample, in Formula 13, [n′/(n′+n″)]×100% is preferably from about 5% toabout 15%.

Alternatively, the transition metal complex can have one or morereactive group(s) for immobilization or conjugation of the complexes toother substrates or carriers, examples of which include, but are notlimited to, macromolecules (e.g., enzymes) and surfaces (e.g., electrodesurfaces).

For reactive attachment to polymers, substrates, or other carriers, thetransition metal complex precursor includes at least one reactive groupthat reacts with a reactive group on the polymer, substrate, or carrier.Typically, covalent bonds are formed between the two reactive groups togenerate a linkage. Examples of such reactive groups and resultinglinkages are provided in Table 1 below. Generally, one of the reactivegroups is an electrophile and the other reactive group is a nucleophile.

TABLE 1 Examples of Reactive Groups and Resulting Linkages FirstReactive Group Second Reactive Group Resulting Linkage Activated ester*Amine Carboxamide Acrylamide Thiol Thioether Acyl azide AmineCarboxamide Acyl halide Amine Carboxamide Carboxylic acid AmineCarboxamide Aldehyde or ketone Hydrazine Hydrazone Aldehyde or ketoneHydroxyamine Oxime Alkyl halide Amine Alkylamine Alkyl halide Carboxylicacid Carboxylic ester Alkyl halide Imidazole Imidazolium Alkyl halidePyridine Pyridinium Alkyl halide Alcohol/phenol Ether Alkyl halide ThiolThioether Alkyl sulfonate Thiol Thioether Alkyl sulfonate PyridinePyridinium Alkyl sulfonate Imidazole Imidazolium Alkyl sulfonateAlcohol/phenol Ether Anhydride Alcohol/phenol Ester Anhydride AmineCarboxamide Aziridine Thiol Thioether Aziridine Amine AlkylamineAziridine Pyridine Pyridinium Epoxide Thiol Thioether Epoxide AmineAlkylamine Epoxide Pyridine Pyridinium Halotriazine Amine AminotriazineHalotriazine Alcohol Triazinyl ether Imido ester Amine AmidineIsocyanate Amine Urea Isocyanate Alcohol Urethane Isothiocyanate AmineThiourea Maleimide Thiol Thioether Sulfonyl halide Amine Sulfonamide*Activated esters, as understood in the art, generally include esters ofsuccinimidyl, benzotriazolyl, or aryl substituted byelectron-withdrawing groups such as sulfo, nitro, cyano, or halo; orcarboxylic acids activated by carbodiimides.

Transition metal complexes of the present invention can be soluble inwater or other aqueous solutions, or in organic solvents. In general,the transition metal complexes can be made soluble in either aqueous ororganic solvents by having an appropriate counter ion or ions, X. Forexample, transition metal complexes with small counter anions, such asF—, Cl—, and Br—, tend to be water soluble. On the other hand,transition metal complexes with bulky counter anions, such as I—, BF₄—and PF₆—, tend to be soluble in organic solvents. Preferably, thesolubility of transition metal complexes of the present invention isgreater than about 0.1 M (moles/liter) at 25° C. for a desired solvent.

The transition metal complexes discussed above are useful as redoxmediators in electrochemical sensors for the detection of analytes inbiofluids. The use of transition metal complexes as redox mediators isdescribed, for example, in U.S. Pat. Nos. 5,262,035, 5,320,725,5,365,786, 5,593,852, 5,665,222, 5,972,199, 6,134,161, 6,143,164,6,175,752 and 6,338,790 and U.S. patent application Ser. No. 09/434,026,all of which are incorporated herein by reference. The transition metalcomplexes described herein can typically be used in place of thosediscussed in the references listed above, although the results of suchuse will be significantly enhanced given the particular properties ofthe transition metal complexes of the present invention, as furtherdescribed herein.

In general, the redox mediator of the present invention is disposed onor in proximity to (e.g., in a solution surrounding) a workingelectrode. The redox mediator transfers electrons between the workingelectrode and an analyte. In some preferred embodiments, an enzyme isalso included to facilitate the transfer. For example, the redoxmediator transfers electrons between the working electrode and glucose(typically via an enzyme) in an enzyme-catalyzed reaction of glucose.Redox polymers are particularly useful for forming non-leachablecoatings on the working electrode. These can be formed, for example, bycrosslinking the redox polymer on the working electrode, or bycrosslinking the redox polymer and the enzyme on the working electrode.

Transition metal complexes can enable accurate, reproducible and quickor continuous assays. Transition metal complex redox mediators acceptelectrons from, or transfer electrons to, enzymes or analytes at a highrate and also exchange electrons rapidly with an electrode. Typically,the rate of self exchange, the process in which a reduced redox mediatortransfers an electron to an oxidized redox mediator, is rapid. At adefined redox mediator concentration, this provides for more rapidtransport of electrons between the enzyme (or analyte) and electrode,and thereby shortens the response time of the sensor. Additionally, thenovel transition metal complex redox mediators are typically stableunder ambient light and at the temperatures encountered in use, storageand transportation. Preferably, the transition metal complex redoxmediators do not undergo chemical change, other than oxidation andreduction, in the period of use or under the conditions of storage,though the redox mediators can be designed to be activated by reacting,for example, with water or the analyte.

The transition metal complex can be used as a redox mediator incombination with a redox enzyme to electrooxidize or electroreduce theanalyte or a compound derived of the analyte, for example by hydrolysisof the analyte. The redox potentials of the redox mediators aregenerally more positive (i.e. more oxidizing) than the redox potentialsof the redox enzymes when the analyte is electrooxidized and morenegative when the analyte is electroreduced. For example, the redoxpotentials of the preferred transition metal complex redox mediatorsused for electrooxidizing glucose with glucose oxidase or PQQ-glucosedehydrogenase as enzyme is between about 200 mV and +200 mV versus aAg/AgCl reference electrode, and the most preferred mediators have redoxpotentials between about −200 mV and about +100 mV versus a Ag/AgClreference electrode.

EXAMPLES OF SYNTHESES OF TRANSITION METAL COMPLEXES

Examples showing the syntheses of various transition metal complexesthat are useful as redox mediators are provided below. Unless indicatedotherwise, all of the chemical reagents are available from AldrichChemical Co. (Milwaukee, Wis.) or other sources. Numerical figuresprovided are approximate.

Example 1 Synthesis of [Os(PY-MIM)₂(MIM)Cl]²⁺2Cl—

By way of illustration, an example of the synthesis of[Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—, as illustrated below, is now provided.

Synthesis of 2-(2-pyridyl)imidazole

A solution of pyridine-2-carboxaldehyde (151.4 g, 1.41 moles) andglyoxal (40% in H₂O, 205 mL, 1.79 moles) in 300 mL of ethanol (EtOH) ina three-necked 1 L round-bottom flask fitted with a thermometer and anaddition funnel was stirred in an ice bath. When the solution was cooledto below 5° C., concentrated NH₄OH (28-30%, 482 mL, 3.93 moles) wasadded dropwise through the addition funnel. The rate of the addition wascontrolled so that the temperature of the solution was maintained atbelow 5° C. After the addition, the stirring of the solution wascontinued in the ice bath for one hour and then at room temperatureovernight. During the stirring process, the solution changed from lightyellow to dark brown.

The solution was transferred to a 2 L round bottom flask and the EtOHsolvent was removed by rotary evaporation. The resulting dark viscousmaterial was transferred to a 4 L beaker with 700 mL of EtOAc. 500 mL ofsaturated NaCl was added and the mixture was stirred for 2 hours. Thesolution was poured into a 2 L separation funnel and a dark tarrymaterial was discarded. The organic layer was separated from thesolution and the aqueous layer was extracted several times with EtOAc(500 mL EtOAc per extraction). The organic layer was then dried withanhydrous Na₂SO₄ overnight, whereupon the resulting mixture was gravityfiltered, the Na₂SO₄ was washed with EtOAc (4×50 mL), and the solutionwas concentrated to about 300-400 mL by rotary evaporation. Theconcentrated solution was transferred to a 1 L Erlenmeyer flask and thevolume was adjusted with more EtOAc to about 400-500 mL, as necessary.The solution stood at 4° C. for 1-2 days to form large amber crystals.The crystals were collected by suction filtration and washed with coldEtOAc (20-30 mL). The filtrate contained a large amount of product, sofurther concentration and crystallization procedures were performed. Thecrystals were combined and dried at 40-45° C. under high vacuum for 2days. The yield of 2-(2-pyridyl)imidazole was about 75 g.

Synthesis of 1-methyl-2-(2-pyridyl)imidazole

Pyridine-2-carboxaldehyde (50.5 g, 0.47 moles) and glyoxal (40% in H₂O,68.3 mL, 0.60 moles) in 100-150 mL of ethanol (EtOH) in a three-necked 1L round-bottom flask fitted with a thermometer and an addition funnelwere stirred in an ice bath. When the solution was cooled to below 5°C., concentrated NH₄OH (28-30%, 161 mL, 1.31 moles) was added dropwisethrough the addition funnel. The rate of the addition was controlled sothat the temperature of the solution was maintained at below 5° C. Afterthe addition, the stirring of the solution was continued in the ice bathfor one hour and then at room temperature overnight. During the stirringprocess, the solution changed from light yellow to dark brown.

The solution was transferred to a 1 L round bottom flask and the EtOHand H₂O solvent was removed by rotary evaporation at 50° C. Theresulting material was dried further at about 50° C. under high vacuumfor 24 hours and then dissolved in anhydrous dimethyl formamide (DMF),whereupon the solution was transferred with further DMF (total DMF450-500 mL) to a three-necked 1 L round bottom flask equipped with areflux condenser, and then stirred. Sodium t-butoxide (48.9 g, 0.51moles) was added quickly via a funnel to obtain, with continued stirringfor about 1 hour, a dark brown homogeneous solution. Methyl iodide (34.5mL, 0.56 moles) was then added dropwise via an addition funnel over1.5-2 hours, resulting in a white precipitate of NaI. The mixture wasstirred at room temperature overnight, its color changing from darkbrown to light brown. The mixture was then poured into a beakercontaining 1.5 mL of EtOAc and suction-filtered using a Buchner funnelto remove the NaI precipitate. The precipitate was washed withadditional EtOAc (3×100 mL). The filtrate was transferred to a 2 L roundbottom flash and rotary evaporated to remove the EtOAc.

The resulting viscous material was transferred to a 1 L beaker with aminimum amount of EtOAc, which was then removed by rotary evaporation.The remaining DMF was removed by vacuum distillation using a low vacuumdiaphragm pump and an oil bath. Upon complete removal of the DMF, theproduct was distilled at 100-110° C. under high vacuum. The yield of1-methyl-2-(2-pyridyl)imidazole was about 36 g.

Synthesis of Os(PY-MIM)₂Cl₂

1-methyl-2-(2-pyridyl)imidazole (3.4 g, 21.4 mmoles) and ammoniumhexachloroosmiate (IV) (4.7 g, 10.7 mmoles) were combined with anhydrousethylene glycol (86 mL) in a three-necked 250 mL round bottom flask,fitted with a reflux condenser, immersed in a temperature-controlled oilbath. The reaction mixture was degassed with N₂ for about 15 minutes.The mixture was stirred under N₂ while the heater was turned on to heatthe oil bath, and the reaction proceeded at 130° C. for 2 hours andsubsequently at 140° C. for about 28 hours until an intermediate thatwas formed in the reaction was completely converted to the finalproduct. The solution was cooled to room temperature and thensuction-filtered through a fritted funnel into a three-necked 250 mLround bottom flask, whereupon a small amount of orange precipitate leftin the funnel was discarded. The solution (solution A) was then degassedwith N₂ for 15 minutes and kept under N₂.

Deionized H₂O (320 mL) was then degassed with N₂ in a three-necked 500mL round bottom flask cooled in an ice/water bath and equipped with athermometer. After 15 minutes of degassing, sodium hydrosulfite (85%,9.31 g, 53.5 mmoles) under N₂ was added immediately and degassingcontinued for another 10-15 minutes. The temperature of the solution(solution B) was below 5° C. Solution A was then added via a canula tosolution B under rapid stirring for about 0.5 hour to form a fine darkpurple precipitate of Os(Py-MIM)₂C12. Stirring continued under N₂ foranother 0.5 hour. The resulting suspension was suction-filtered througha 0.4 or 0.3 micron Nylon membrane. The suspension was transferred tothe suction funnel via a canula under nitrogen to minimize air exposure.The dark purple precipitate was then washed with a minimum of ice coldwater (2×5 mL). The precipitate was immediately dried by lyophilizationfor at least 24 hours. The yield of Os(Py-MIM)₂Cl₂ was about 5.6 g.

Synthesis of [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—

Anhydrous ethanol (1 L) in a 2-L three-necked round bottom flask fittedwith a reflux condenser was degassed with N₂ for 15 minutes.Os(Py-MIM)₂Cl₂ (3.1 g, 5.35 mmoles) was added quickly under N₂ via afunnel. The suspension was stirred and heated to reflux.1-methylimidazole (0.43 mL, 5.35 mmoles) was then added at once via asyringe. Reflux continued until the reaction was completed. During thereaction, the solution changed from dark brown to purple-brown. Thesolution was cooled to room temperature and then suction-filteredthrough a fritted funnel. The solvent was then removed by rotaryevaporation to give the crude product in its reduced form.

The product was transferred with 30-50 mL H₂O to a 400 mL beakercontaining about 40 mL AG1×4 chloride resin from Bio-Rad, or preferably,80 mL Dowex-1-chloride from Aldrich. The mixture was stirred in open airfor about 24 hours to convert Os(II) to Os(III). The mixture was thensuction-filtered and the resin was washed with H₂O (5×30 mL). Thecombined filtrate was concentrated to about 50 mL by rotary evaporationat 35° C. under vacuum.

The solution was loaded onto a LH-20 column (2″×22″), which was elutedwith H₂O. 50 mL fractions were collected and analyzed by CV to find themajor purple-brown band associated with the product. Fractionscontaining pure product were collected and concentrated by rotaryevaporation to about 150 mL. The solution was then freeze-dried to givethe product. The yield of [Os(Py-MIM)₂)Cl]²⁺2Cl— was about 2.4 g.

As described herein, [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl— is a transition metalcomplex that is particularly useful as a redox mediator.

Example 2 Synthesis of 1-phenyl-2-(2-pyridyl)imidazole

Further by way of illustration, an example of the synthesis of1-phenyl-2-(2-pyridyl)imidazole, as illustrated below, is now provided.The example demonstrates how a 1-aryl-substituted 2-(2-pyridyl)imidazoleis made from 1-(2-pyridyl)imidazole or its derivative, and aniodobenzene derivative (as illustrated) or a bromobenzene derivative.

2-(2-pyridyl)imidazole (6.91 g), iodobenzene (11.47 g), Cs₂CO₃ (25 g),and copper powder (15 g) were mixed in 60 mL anhydrous DMF in a 250 mLround bottom flask equipped with a magnetic stirrer and a refluxcondenser. The mixture is degassed with N₂ for 15 minutes at roomtemperature and then refluxed under N₂ in an oil bath for 24 hours. Theresulting mixture was cooled to room temperature and suction-filtered toremove the solid byproduct. The filtrate was extracted with EtOAc (3×100mL). The combined organic layer was washed with H₂O (2×100 mL) and thenwith saturated NaCl (2×150 mL), and subsequently dried with anhydrousNa₂SO₄. Evaporation of the solvent gave crude1-phenyl-2(2-pyridyl)imidazole. The crude product is generally pureenough to use in making redox mediators, although the crude product maybe further purified using a silica gel column and eluting withMeOH/CHCl₃.

The 1-phenyl-2-(2-pyridyl)imidazole product described above can be usedin the synthesis of a transition metal complex, such as an Osmiumcomplex, in much the same manner 1-methyl-2-(2-pyridyl)imidazole wasused in Example 1 above.

Examples of Further Transition Metal Complexes

Further transition metal complexes that serve as redox mediatorsaccording to the present invention are provided in Table 2 below, asMediator Nos. 1-13. The redox potentials (E_(1/2) (mV) relative to astandard Ag/AgCl reference electrode in a pH 7 PBS buffer) associatedwith these redox mediators are also provided, where available.

Also provided in Tables 3 and 4 are various of these redox mediators andtheir associated redox potentials and associated slopes, k, ofsubstantially linear plots of collected charge (μC) versus glucoseconcentration (mg/dL) for a given volume (˜315 ηL) of biofluid, such asblood, as further described below. Comparative information for knownredox mediators, namely, Comparative Mediator Nos. I, X, XII and XIII isalso provided. The slope data in Table 3 and Table 4 concerns redoxmediators tested under Condition A and Condition B, respectively, whichreflect different ink lots, as now described.

That is, these slope data were obtained from individual tests in whicheach mediator and an enzyme mixture were coated on a working electrode.The working electrode was made of a conductive ink layered over aplastic substrate. The working electrode was laminated together with acounter/reference electrode, using standard processing known in the art.The counter/reference electrode was made of a Ag/AgCl ink layered over aplastic substrate. Variations are routinely observed in test stripsensors made from different ink lots. Thus, in Table 3, Condition Arefers to tests conducted using a series of test strips made from asingle lot, and in Table 4, Condition B similarly refers to testsconducted using a series of test strips made from a single lot,different from that associated with Condition A. Thus, comparisons ofslope data shown in Table 3 and Table 4 should not be made, whilecomparisons of slope data shown within either Table 3 or Table 4 areinstructive as to mediator performance.

TABLE 2 Examples of Low Potential Mediators of the Present InventionRedox Potential, E_(½) (mV) Mediator No. Structure of Mediator versusAg/AgCl 1

−164 2

−168 3

−150 4

−172 5

6

7

8

−139 9

−124 10

−117 11

−130 12

−166 13

−88

TABLE 3 Examples of Low Potential Osmium Mediators and Known ComparativeMediators and Properties Thereof Under Condition A Mediator No. RedoxPotential, or Comparative Structure of Mediator E_(½) (mV) Linear Slope,k Mediator No. or Comparative Mediator versus Ag/AgCl (μC/(mg/dL)) 1

−164 1.52 2

−168 1.49 3

−150 1.46 4

−172 1.49 11

−130 1.55 I*

−110 1.14 X*

−125 1.05 *These known comparative mediators are disclosed inInternational Publication No. WO 01/36430 A1 and are merely comparativeexamples herein.

TABLE 4 Examples of Low Potential Osmium Mediators and Known ComparativeMediators and Properties Thereof Under Condition B Mediator No. RedoxPotential, or Comparative Structure of Mediator E_(½) (mV) Linear Slope,k Mediator No. or Comparative Mediator Versus Ag/AgCl) (μC(mg/dL)) 8

−139 1.73 9

−124 1.70 X*

−125 1.48 XII*

−74 1.46 XIII*

−97 1.52 *These known comparative mediators are disclosed inInternational Publication No. WO 01/36430 A1 and are merely comparativeexamples herein.

The transition metal complexes of the present invention are well suitedfor electrochemical sensing applications, given their particularelectrochemical properties. For example, as shown above, the redoxpotentials of the mediators are generally low, such as in a range offrom about 0 mV to about −200 mV relative to a Ag/AgCl referenceelectrode. These redox potentials are particularly desirable forelectrochemical sensing applications, being in a range at which thekinetics of the mediators is fast and the electrochemical activity ofpotentially interfering species is minimized. Mediator Nos. 1-13 thusexemplify electrochemically desirable mediators according to the presentinvention.

The identity of the potentially interfering species, just describeddepends on the particular electrochemical sensing application. Merely byway of example, when the electrochemical sensing application concernsthe biofluid, blood, potentially interfering species include ascorbicacid, acetaminophen, and uric acid. Mediator Nos. 1-13 exemplifyelectrochemically desirable mediators that operate at potentialssuitable for minimizing the electrochemical activity of such potentiallyinterfering species, while not sacrificing mediator efficiency.

Additionally, the transition metal complexes of the present inventionare particularly effective redox mediators in electrochemical sensingapplications, given their enhanced ability to collect charge at theworking electrode, which in turn enhances the sensitivity of the sensorto the concentration of the analyte being sensed. By way of example, inthe general operation of an electrochemical biosensor, such as a glucosesensor, the reduced enzyme, glucose oxidase or glucose dehydrogenase,transfers its electrons to the working electrode via a particularprocess. In that process, the oxidized form of the redox mediatorinteracts with the reduced enzyme, thereby receiving an electron andbecoming reduced. The reduced mediator travels to the surface of theworking electrode, typically by random diffusion, whereupon it transfersthe collected electron to the electrode, thereby becoming oxidized.

Ideally, because each glucose molecule loses two electrons in theabove-described process, the total amount of electrons or chargecollected at the working electrode should be equal to two times thenumber of glucose molecules oxidized. In practice, however, the totalamount of charge collected is almost always less than the ideal ortheoretical amount because the electrons may be “lost” during transferfrom the enzyme to the electrode. For example, the reduced enzyme maytransfer the electrons to oxygen or other chemical species, rather thanto the redox mediator. An efficient redox mediator should thus competefavorably for electrons from the enzyme.

Further, ideally, once the redox mediator receives an electron from theenzyme, it should not transfer the electron to another oxidativespecies, such as oxygen or other chemicals present in the sensor, beforebeing oxidized on the working electrode. A good mediator should thuscompete favorably for electrons from the reduced enzyme, as describedabove, and be substantially chemically inert during its random diffusionto the working electrode whereupon it is oxidized.

An efficient mediator is particularly important in coulometry-basedelectrochemical biosensing, in which detection of the bioanalyte isbased on the total amount of charge collected at the working electrodefor a given volume of biofluid. When greater charge is collected at theworking electrode, the sensor is advantageously more sensitive. For acoulometry-based glucose sensor, for example, the sensitivity of thesensor may be characterized by the slope value of a linear plot ofcharge versus glucose concentration as defined by the equation y=kx+b,where y is the collected charge in μC for a given volume of biofluid, kis the slope in μC/(mg/dL), x is the glucose concentration in mg/dL, andb is the intercept based on background charge. As demonstrated above,mediators of the present invention that have a negatively chargedligand, such as Mediator Nos. 1-13 that have a chloride ligand, haveassociated slope values that are significantly higher (for example,about 28% to about 48% higher per Table 3, and about 11% to about 18%higher per Table 4) than those of mediators that have heterocyclicnitrogen-containing ligands surrounding the metal redox center, asexemplified by Comparative Mediator Nos. I, X, XII and XIII.

The above-described data demonstrate favorable properties of transitionmetal complexes that make these complexes particularly desirable redoxmediators. In electrochemical sensing applications, such as theelectrochemical sensing of glucose, the transition metal complexeseffectively collect electrons from the reduced enzyme and effectivelyretain the collected electrons prior to delivering them to the workingelectrode.

As described herein, the transition metal complexes of the presentinvention are usefully employed as redox mediators in electrochemicalsensors. These mediators have very fast kinetics, such that electronexchange between such a mediator and the enzyme and/or the workingelectrode in the sensor device is rapid, and more particularly, rapidenough to facilitate the transfer of electrons to the working electrodethat might otherwise be transferred to another electron scavenger, suchas oxygen. The electron-transfer efficiency of a mediator of Formula 1is enhanced when L₂ is a negatively charged ligand, such as a chlorideligand, as demonstrated by the desirable slope values, k, listed abovefor Mediator Nos. 1-13. By way of comparison, a mediator having aneutral ligand, L₂, such as a heterocyclic nitrogen-containing ligand,is less able to transfer electrons from the enzyme to the workingelectrode, as reflected by the lower slope values listed above forComparative Mediator Nos. I, X, XII and XIII.

The transition metal complex mediators of the present invention are alsoquite stable in terms of chemical reactivity with respect to chemicalspecies other than the enzyme and the electrode surface. By way ofexample, the chemical stability of a mediator of the present inventionis such that preferably the predominant, or most preferably the only,reactions in which it participates involves the above-described,electron-transfer reaction between the mediator and the enzyme and theelectrochemical redox reaction at the working electrode. This chemicalstability may be enhanced when a mediator of Formula 1, wherein L₂ is anegatively charged monodentate ligand, has a “bulky” chemical ligand,L₁, that spatially or stereochemically shields the redox center, such asOs^(2+/3+), and thereby, reduces undesirable chemical reactivity beyondthe fundamentally desired chemical and electrochemical activity.Mediator Nos. 1-13, above, are particular examples of such “bulked”,chemically stable mediators of the present invention.

Further by way of example, the thermal and photochemical stability of amediator of the present invention is preferably such that the mediatoris temperature- and light-stable, respectively, under typical use,storage and transportation conditions. For example, mediators of thepresent invention may be easily handled under normal lighting conditionsand may have a shelf life of at least about 18 months at about roomtemperature, and at least about 2 weeks at about 57° C. Mediator Nos.1-13, above, are particular examples of such thermally andphotochemically stable mediators of the present invention.

Mediators of the present invention have desirable redox potentials in arange at which the electron-transfer kinetics is optimized, ormaximized, and the effect of common interfering species present inbiofluid is minimized. Mediator Nos. 1-13, above, are particularexamples of mediators of suitable redox potential.

The transition metal complex mediators of the present invention alsohave desirable solubility properties, generally having a solubility ofgreater than about 0.1 moles/liter at 25° C. for a desired solvent,which is typically an aqueous or a water-miscible solvent.Advantageously, one need only adjust the counter ion or ions, X, ofFormula 1, to obtain a desirable solubility for the solvent of choice,be it aqueous or organic.

In summary, the present invention provides novel transition metalcomplexes that are particularly useful as redox mediators inelectrochemical sensing applications. The preferred redox mediatorsexchange electrons rapidly with enzymes and working electrodes, arestable, are readily synthesized, and have redox potentials that aretailored for the electrooxidation of a variety of analytes, such asthose in various biological fluids within the human body. Whilemediators of the present invention have been described for the most partin terms of glucose sensing, they are useful for the sensing of otheranalytes, such as lactic acid for example. Generally, if the redoxpotential of the enzyme used in a particular analyte-sensing applicationis negative relative to the redox potential of the mediator, themediator is suitable for that analyte-sensing application. Theadvantageous properties and characteristics of the transition metalcomplexes of the present invention make them ideal candidates for use inthe electrochemical sensing of glucose, an application of particularimportance in the diagnosis and monitoring of diabetes in humanpopulations.

Various aspects and features of the present invention have beenexplained or described in relation to beliefs or theories, although itwill be understood that the invention is not bound to any belief ortheory. Further, various modifications, equivalent processes, as well asnumerous structures to which the present invention may be applicablewill be readily apparent to those of skill in the art to which thepresent invention is directed upon review of the instant specification.Although the various aspects and features of the present invention havebeen described with respect to various embodiments and specific examplesherein, it will be understood that the invention is entitled toprotection within the full scope of the appended claims.

1. A test strip comprising: a working electrode; a counter electrode;wherein the working electrode comprises an analyte-responsive enzyme anda mediator complex, the mediator complex having the formula:

wherein c is a negative, neutral, or positive charge represented by −1to −5, 0, or +1 to +5, inclusive, respectively; d is a number of counterions, X, from 0 to 5, inclusive; M is osmium or ruthenium; L₁ is amethylamino substituted pyridine; L₂ is a negatively charged ligand; andL and L′ are independently:

wherein R′₁ is a substituted or an unsubstituted alkyl, alkenyl or aryl;each of R_(a) and R_(b) is independently —H, —F, —Cl, —Br, —I, —NO₂,—CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted orunsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido,hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio,alkenyl, aryl, or alkyl; each of R_(e) and R_(d) is independently —H,—F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, orsubstituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino,alkylthio, alkenyl, aryl, or alkyl, or a combination of R_(e) and R_(d)forms a saturated or an unsaturated 5- or 6-membered ring; and each ofR′₃ and R′₄ is independently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H,—SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted or unsubstitutedalkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl,or alkyl, or a combination of R′₃ and R′₄ forms a saturated or anunsaturated 5- or 6-membered ring.
 2. The test strip of claim 1, whereinM is osmium; L₂ is a halide; and L and L′ are independently:

wherein R′₁ is a substituted or an unsubstituted C1-C6 alkyl; R′₃ andR′₄ are independently —H; R_(a), R_(b), R_(c) and R_(d) areindependently —H or C1 alkyl; c is a negative, neutral, or positivecharge represented by −1 to −5, 0, or +1 to +5, inclusive, respectively.3. The test strip of claim 2, wherein L₁ has the formula:

wherein one of R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is methylamino.
 4. The teststrip of claim 3, wherein R₁₁ and R₁₅ are independently —H; R₁₂ and R₁₄are the same and —H; and R₁₃ is methylamino.
 5. The test strip of claim2, wherein L₂ is —F, —Cl, or —Br.
 6. The test strip of claim 2, whereinL₂ is —Cl.
 7. The test strip of claim 2, wherein R′₁ is a C1-C2 alkyl.8. The test strip of claim 2, wherein R′₁ is a C1 alkyl.
 9. The teststrip of claim 2, wherein each of R_(a) and R_(c) is —H.
 10. The teststrip of claim 2, wherein each of R_(a), R_(b), and R_(c) is —H.
 11. Thetest strip of claim 2, wherein each of R_(a), R_(b), R_(c), and R_(d) is—H.
 12. The test strip of claim 2, wherein X is a halide.
 13. The teststrip of claim 2, wherein X is chloride.
 14. The test strip of claim 1,wherein the analyte-responsive enzyme is glucose dehydrogenase (GDH).15. The test strip of claim 1, wherein the analyte-responsive enzymefurther comprises an enzyme cofactor.
 16. A test strip comprising: aworking electrode; a counter electrode; wherein the working electrodecomprises an analyte-responsive enzyme and a mediator complex, themediator complex having the formula:

wherein M is cobalt, iron, osmium, ruthenium, or vanadium; L₁ has theformula:

wherein R₁₁, R₁₂, R₁₄ and R₁₅ are —H and R₁₃ is methlyamino; L₂ ischloride; L and L′ are independently:

wherein R′₁ is methyl; and R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) areindependently —H; c is a negative, neutral, or positive chargerepresented by −1 to −5, 0, or +1 to +5, inclusive, respectively; d is anumber of X sufficient to balance the charge c; and X is an anion. 17.The test strip of claim 16, wherein the analyte-responsive enzyme isglucose dehydrogenase (GDH).
 18. The test strip of claim 16, wherein theanalyte-responsive enzyme further comprises an enzyme cofactor.
 19. Atest strip comprising: a working electrode; a counter electrode; whereinthe working electrode comprises an analyte-responsive enzyme and amediator complex, the mediator complex having the formula:

wherein M is osmium; L₁ has the formula:

wherein R₁₁, R₁₂, R₁₄ and R₁₅ are —H; R₁₃ is methylamino; L₂ ischloride; L and L′ are independently:

wherein R′₁ methyl; and R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) areindependently —H; c is +2; d is 2; and X is chloride.
 20. The test stripof claim 19, wherein the analyte-responsive enzyme is glucosedehydrogenase (GDH).
 21. The test strip of claim 19, wherein theanalyte-responsive enzyme further comprises an enzyme cofactor.